Role for Carboxylic Acid Moiety in NSAIDs: Favoring the Binding at Site II of Bovine Serum Albumin.

The molecular structures of nonsteroidal anti-inflammatory drugs (NSAIDs) vary, but most contain a carboxylic acid functional group (RCOOH). This functional group is known to be related to the mechanism of cyclooxygenase inhibition and also causes side effects, such as gastrointestinal bleeding. This study proposes a new role for RCOOH in NSAIDs: facilitating the interaction at the binding site II of serum albumins. We used bovine serum albumin (BSA) as a model to investigate the interactions with ligands at site II. Using dansyl-proline (DP) as a fluorescent site II marker, we demonstrated that only negatively charged NSAIDs such as ibuprofen (IBP), naproxen (NPX), diflunisal (DFS), and ketoprofen (KTP) can efficiently displace DP from the albumin binding site. We confirmed the importance of RCOO by neutralizing IBP and NPX through esterification, which reduced the displacement of DP. The competition was also monitored by stopped-flow experiments. While IBP and NPX displaced DP in less than 1 s, the ester derivatives were ineffective. We also observed a higher affinity of negatively charged NSAIDs using DFS as a probe and ultrafiltration experiments. Molecular docking simulations showed an essential salt bridge between the positively charged residues Arg409 and Lys413 with RCOO-, consistent with the experimental findings. We performed a ligand dissociation pathway and corresponding energy analysis by applying molecular dynamics. The dissociation of NPX showed a higher free energy barrier than its ester. Apart from BSA, we conducted some experimental studies with human serum albumin, and similar results were obtained, suggesting a general effect for other mammalian serum albumins. Our findings support that the RCOOH moiety affects not only the mechanism of action and side effects but also the pharmacokinetics of NSAIDs.

Elucidation of binding mechanisms of bovine serum albumin and 1-alkylsulfonates with different hydrophobic chain lengths.

In this article, the binding interactions between bovine serum albumin (BSA) and three 1-alkylsulfonates, namely sodium 1-dodecanesulfonate, sodium 1-decanesulfonate, and sodium 1-octanesulfonate, have been thoroughly investigated. The study employed various experimental techniques such as isothermal titration calorimetry (ITC), steady-state fluorescence spectroscopy (SF), circular dichroism spectroscopy (CD), and molecular dynamics-based simulations. The objective was to understand the influence of the alkyl chain length of the investigated ligands on several aspects, including the strength of the interaction, the stoichiometry of the resulting complexes, the number of BSA binding sites, and the underlying mechanisms of binding. Notably, the study also demonstrated that sodium dodecyl sulfate (S12S) can serve as an effective site marker for BSA when studying ligands with similar structural and topological features. These findings may have significant implications for enhancing our understanding of the interactions between small amphiphilic molecules and proteins.

Benchmarking Water Models in Molecular Dynamics of Protein-Glycosaminoglycan Complexes.

Glycosaminoglycans (GAGs) made of repeating disaccharide units intricately engage with proteins, playing a crucial role in the spatial organization of the extracellular matrix (ECM) and the transduction of biological signals in cells to modulate a number of biochemical processes. Exploring protein-GAG interactions reveals several challenges for their analysis, namely, the highly charged and periodic nature of GAGs, their multipose binding, and the abundance of the interfacial water molecules in the protein-GAG complexes. Most of the studies on protein-GAG interactions are conducted using the TIP3P water model, and there are no data on the effect of various water models on the results obtained in molecular dynamics (MD) simulations of protein-GAG complexes. Hence, it is essential to perform a systematic analysis of different water models in MD simulations for these systems. In this work, we aim to evaluate the properties of the protein-GAG complexes in MD simulations using different explicit: TIP3P, SPC/E, TIP4P, TIP4PEw, OPC, and TIP5P and implicit: IGB = 1, 2, 5, 7, and 8 water models to find out which of them are best suited to study the dynamics of protein-GAG complexes. The FF14SB and GLYCAM06 force fields were used for the proteins and GAGs, respectively. The interactions of several GAG types, such as heparin, chondroitin sulfate, and hyaluronic acid with basic fibroblast growth factor, cathepsin K, and CD44 receptor, respectively, are investigated. The observed variations in different descriptors used to study the binding in these complexes emphasize the relevance of the choice of water models for the MD simulation of these complexes.

Detecting Protein-Ligand Interactions with Nitroxide Based Paramagnetic Cosolutes.

NMR spectroscopy techniques can provide important information about protein-ligand interactions. Here we tested an NMR approach which relies on the measurement of paramagnetic relaxation enhancements (PREs) arising from analogous cationic, anionic or neutral soluble nitroxide molecules, which distribute around the protein-ligand complex depending on near-surface electrostatic potentials. We applied this approach to two protein-ligand systems, interleukin-8 interacting with highly charged glycosaminoglycans and the SH2 domain of Grb2 interacting with less charged phospho-tyrosine tripeptides. The electrostatic potential around interleukin-8 and its changes upon binding of glycosaminoglycans could be derived from the PRE data and confirmed by theoretical predictions from Poisson-Boltzmann calculations. The ligand influence on the PREs and NMR-derived electrostatic potentials of Grb2 SH2 was localized to a narrow protein region which allowed the localization of the peptide binding pocket. Our analysis suggests that experiments with nitroxide cosolutes can be useful for investigating protein-ligand electrostatic interactions and mapping ligand binding sites.

Ligand binding of interleukin-8: a comparison of glycosaminoglycans and acidic peptides.

Recognition and binding of regulatory proteins to glycosaminoglycans (GAGs) from the extracellular matrix is a process of high biological importance. The interaction between negatively charged sulfate or carboxyl groups of the GAGs and clusters of basic amino acids on the protein is crucial in this binding process and it is believed that electrostatics represent the key factor for this interaction. However, given the rather undirected nature of electrostatics, it is important to achieve a clear understanding of its role in protein-GAG interactions and how specificity and selectivity in these systems can be achieved, when the classical key-lock binding motif is not applicable. Here, we compare protein binding of a highly charged heparin (HP) hexasaccharide with four de novo designed decapeptides of varying negative net charge. The charge density of these peptides was comparable to typical GAGs of the extracellular matrix. We used the regulatory protein interleukin-8 (IL-8) because its interactions with GAGs are well described. All four peptide ligands bind to the same epitope of IL-8 but show much weaker binding affinity as revealed in 1H-15N HSQC NMR titration experiments. Complementary molecular docking and molecular dynamics simulations revealed further atomistic details of the interaction mode of GAG versus peptide ligands. Overall, similar contributions to the binding energy and hydrogen bond formation are determined for HP and the highly charged peptides, suggesting that the entropic loss of the peptides upon binding likely account for the remarkably different affinity of GAG versus peptide ligands to IL-8.

Extension of the SUGRES-1P Coarse-Grained Model of Polysaccharides to Heparin.

Heparin is an unbranched periodic polysaccharide composed of negatively charged monomers and involved in key biological processes, including anticoagulation, angiogenesis, and inflammation. Its structure and dynamics have been studied extensively using experimental as well as theoretical approaches. The conventional approach of computational chemistry applied to the analysis of biomolecules is all-atom molecular dynamics, which captures the interactions of individual atoms by solving Newton's equation of motion. An alternative is molecular dynamics simulations using coarse-grained models of biomacromolecules, which offer a reduction of the representation and consequently enable us to extend the time and size scale of simulations by orders of magnitude. In this work, we extend the UNIfied COarse-gRaiNed (UNICORN) model of biological macromolecules developed in our laboratory to heparin. We carried out extensive tests to estimate the optimal weights of energy terms of the effective energy function as well as the optimal Debye-Hückel screening factor for electrostatic interactions. We applied the model to study unbound heparin molecules of polymerization degree ranging from 6 to 68 residues. We compare the obtained coarse-grained heparin conformations with models obtained from X-ray diffraction studies of heparin. The SUGRES-1P force field was able to accurately predict the general shape and global characteristics of heparin molecules.

Insights into the interaction of human serum albumin with ionic liquids - Thermodynamic, spectroscopic and molecular modelling studies.

Human serum albumin (HSA) effectively binds different types of low-molecular-weight compounds and thus enables their distribution in living organisms. Recently, it has been reported that the protein-ligand interactions play a crucial role in bioaccumulation processes and provide an important sorption phase, especially for ionogenic compounds. Therefore, the binding interactions of such compounds with proteins are the subject of an ongoing interest in environmental and life sciences. In this paper, the influence of some counter-ions, namely [B(CN)4]- and [C(CN)3]- on the affinity of the [IM1-12]+ towards HSA has been investigated and discussed based on experimental methods (isothermal titration calorimetry and steady-state fluorescence spectroscopy) and molecular dynamics-based computational approaches. Furthermore, the thermal stability of the resulting HSA/ligand complexes was assessed using DSC and CD spectroscopy. As an outcome of the work, it has been ascertained that the protein is able to bind simultaneously the ligands under study but in different regions of HSA. Thus, the presence in the system of [IM1-12]+ does not disturb the binding of [C(CN)3]- and [B(CN)4]-. The presented results provide important information on the presence of globular proteins and some ionogenic compounds in the distribution and bioaccumulation of ILs in the environment and living organisms.

Glycosaminoglycans: What Remains To Be Deciphered?

Glycosaminoglycans (GAGs) are complex polysaccharides exhibiting a vast structural diversity and fulfilling various functions mediated by thousands of interactions in the extracellular matrix, at the cell surface, and within the cells where they have been detected in the nucleus. It is known that the chemical groups attached to GAGs and GAG conformations comprise "glycocodes" that are not yet fully deciphered. The molecular context also matters for GAG structures and functions, and the influence of the structure and functions of the proteoglycan core proteins on sulfated GAGs and vice versa warrants further investigation. The lack of dedicated bioinformatic tools for mining GAG data sets contributes to a partial characterization of the structural and functional landscape and interactions of GAGs. These pending issues will benefit from the development of new approaches reviewed here, namely (i) the synthesis of GAG oligosaccharides to build large and diverse GAG libraries, (ii) GAG analysis and sequencing by mass spectrometry (e.g., ion mobility-mass spectrometry), gas-phase infrared spectroscopy, recognition tunnelling nanopores, and molecular modeling to identify bioactive GAG sequences, biophysical methods to investigate binding interfaces, and to expand our knowledge and understanding of glycocodes governing GAG molecular recognition, and (iii) artificial intelligence for in-depth investigation of GAGomic data sets and their integration with proteomics.

Computational modeling of the molecular basis for the calcium-dependence of the mannuronan C-5 epimerase AvAlgE6 from Azotobacter vinelandii.

The mannuronan C-5 epimerases catalyze epimerization of ß-d-mannuronic acid to α-l-guluronic acid in alginate polymers. The seven extracellular Azotobacter vinelandii epimerases (AvAlgE1-7) are calcium-dependent, and calcium is essential for the structural integrity of their carbohydrate binding R-modules. Ca2+ is also found in the crystal structures of the A-modules, where it is suggested to play a structural role. In this study, the structure of the catalytic A-module of the A. vinelandii mannuronan C-5 epimerase AvAlgE6 is used to investigate the role of this Ca2+. Molecular dynamics (MD) simulations with and without calcium reveal the possible importance of the bound Ca2+ in the hydrophobic packing of ß-sheets. In addition, a putative calcium binding site is found in the active site, indicating a potential direct role of this calcium in the catalysis. According to the literature, two of the residues coordinating calcium in this site are essential for the activity. MD simulations of the interaction with bound substrate indicate that the presence of a calcium ion in this binding site increases the binding strength. Further, explicit calculations of the substrate dissociation pathways with umbrella sampling simulations show and energetically higher dissociation barrier when calcium is present. The present study eludes to a putative catalytic role of calcium in the charge neutralizing first step of the enzymatic reaction. In addition to the importance for understanding these enzymes' molecular mechanisms, this could have implications for engineering strategies of the epimerases in industrial alginate processing.

Solvent Model Benchmark for Molecular Dynamics of Glycosaminoglycans.

In computational studies of glycosaminoglycans (GAGs), a group of anionic, periodic linear polysaccharides, so far there has been very little discussion about the role of solvent models in the molecular dynamics simulations of these molecules. Predominantly, the TIP3P water model is commonly used as one of the most popular explicit water models in general. However, there are numerous alternative explicit and implicit water models that are neglected in the computational research of GAGs. Since solvent-mediated interactions are particularly important for GAG dynamic and structural properties, it would be of great interest for the GAG community to establish the solvent model that is suited the best in terms of the quality of theoretically obtained GAG parameters and, at the same time, would be reasonably demanding in terms of computational resources required. In this study, heparin (HP) was simulated using five implicit and six explicit solvent models with the aim to find out how different solvent models influence HP's molecular descriptors in the molecular dynamics simulations. Here, we initiate the search for the most appropriate solvent representation for GAG systems and we hope to encourage other groups to contribute to this highly relevant subject.

Molecular Dynamics-Based Comparative Analysis of Chondroitin and Dermatan Sulfates.

Glycosaminoglycans (GAGs) are a class of linear anionic periodic polysaccharides containing disaccharide repetitive units. These molecules interact with a variety of proteins in the extracellular matrix and so participate in biochemically crucial processes such as cell signalling affecting tissue regeneration as well as the onset of cancer, Alzheimer's or Parkinson's diseases. Due to their flexibility, periodicity and chemical heterogeneity, often termed "sulfation code", GAGs are challenging molecules both for experiments and computation. One of the key questions in the GAG research is the specificity of their intermolecular interactions. In this study, we make a step forward to deciphering the "sulfation code" of chondroitin sulfates-4,6 (CS4, CS6, where the numbers correspond to the position of sulfation in NAcGal residue) and dermatan sulfate (DS), which is different from CSs by the presence of IdoA acid instead of GlcA. We rigorously investigate two sets of these GAGs in dimeric, tetrameric and hexameric forms with molecular dynamics-based descriptors. Our data clearly suggest that CS4, CS6 and DS are substantially different in terms of their structural, conformational and dynamic properties, which contributes to the understanding of how these molecules can be different when they bind proteins, which could have practical implications for the GAG-based drug design strategies in the regenerative medicine.

Investigation of hexacyanoferrate(II)/(III) charge-dependent interactions with bovine and human serum albumins.

In the present paper, the binding interactions of highly negative-charged ions, namely hexacyanoferrates(II/III), i.e. [Fe(CN)6]4- and [Fe(CN)6]3- with bovine and human serum albumins (BSA and HSA, respectively) have been studied for the first time in an aqueous solution (10 mM cacodylate buffer of pH 7.0) using steady-state fluorescence spectroscopy, isothermal titration calorimetry, and CD spectroscopy supported by molecular dynamics-based computational approaches. The Stern-Volmer equation as well as its modifications suggested that hexacyanoferrates(II/III) effectively quenched the intrinsic fluorescence of the albumins through a static mechanism. The proteins under study possess only one binding site on the surface capable of binding one mole of hexacyanoferrates(II/III) ions per one mole of albumin (HSA or BSA). The formation of albumin complexes is an enthalpy-driven process (|ΔHITC| > |TΔSITC|). The strength of the interactions depends mainly on the type of albumin, and changes as follows: BSA-K3[Fe(CN)6] âˆ¼ BSA-K4[Fe(CN)6] > HSA-K3[Fe(CN)6] âˆ¼ HSA-K4[Fe(CN)6]. Finally, potential binding sites of bovine and human serum albumins have been investigated and discussed based on a competitive fluorescence displacement assay (with warfarin and ibuprofen as site markers) and molecular dynamics simulations.

Repulsive Scaling Replica Exchange Molecular Dynamics in Modeling Protein-Glycosaminoglycan Complexes.

Glycosaminoglycans are long linear periodic anionic polysaccharides consisting of disaccharide units exhibiting different sulfation patterns forming a highly heterogeneous group of molecules. Due to their flexibility, length, high charge, and periodicity, they are challenging for computational approaches. Despite their biological significance in terms of the important role in various diseases (e.g., Alzheimer, cancer, SARS-CoV-2) and proper cell functioning (e.g., proliferation, maturation), there is a lack of effective molecular docking tools designed specifically for glycosaminoglycans due to their challenging physical-chemical nature. In this chapter we present protocols for the Repulsive Scaling Replica Exchange Molecular Dynamics (RS-REMD) methods to dock glycosaminoglycans with both implicit and explicit solvent models implemented. This novel molecular dynamics-based replica exchange technique should help to elevate our current knowledge on the complexes and interactions between glycosaminoglycans and their protein receptors.

In silico insights into procathepsin S maturation mediated by glycosaminoglycans.

Procathepsins, inactive precursors of cathepsins are present in the extracellular matrix (ECM) and in lysosomes. Their active forms are involved in a number of biologically relevant processes, including bone resorption, intracellular proteolysis and regulation of programmed cell death. These processes might be mediated by glycosaminoglycans (GAGs), long unbranched periodic negatively charged polysaccharides. GAGs are also present in ECM and play important role in anticoagulation, angiogenesis and tissue regeneration. GAGs not only mediate the enzymatic activity of cathepsins but can also regulate the process of procathepsin maturation, as it was shown for procathepsin B and S. In this study, we propose the molecular mechanism underlying the biological role of GAGs in procathepsin S maturation and compare our findings with computational data obtained for procathepsin B. We rigorously analyse procathepsin S-GAG complexes in terms of their dynamics, free energy and potential allosteric regulation. We conclude that the GAG binding region might have an effect on the dynamics of procathepsin S structure and so affect its maturation by two different mechanisms.

Studying specificity in protein-glycosaminoglycan recognition with umbrella sampling.

In the past few decades, glycosaminoglycan (GAG) research has been crucial for gaining insights into various physiological, pathological, and therapeutic aspects mediated by the direct interactions between the GAG molecules and diverse proteins. The structural and functional heterogeneities of GAGs as well as their ability to bind specific proteins are determined by the sugar composition of the GAG, the size of the GAG chains, and the degree and pattern of sulfation. A deep understanding of the interactions in protein-GAG complexes is essential to explain their biological functions. In this study, the umbrella sampling (US) approach is used to pull away a GAG ligand from the binding site and then pull it back in. We analyze the binding interactions between GAGs of three types (heparin, desulfated heparan sulfate, and chondroitin sulfate) with three different proteins (basic fibroblast growth factor, acidic fibroblast growth factor, and cathepsin K). The main focus of our study was to evaluate whether the US approach is able to reproduce experimentally obtained structures, and how useful it can be for getting a deeper understanding of GAG properties, especially protein recognition specificity and multipose binding. We found that the binding free energy landscape in the proximity of the GAG native binding pose is complex and implies the co-existence of several binding poses. The sliding of a GAG chain along a protein surface could be a potential mechanism of GAG particular sequence recognition by proteins.

Rapamycin-induced autophagy in osteosarcoma cells is mediated via the biglycan/Wnt/ß-catenin signaling axis.

Biglycan is a class I secreted small leucine-rich proteoglycan (SLRP), which regulates signaling pathways connected to bone pathologies. Autophagy is a vital catabolic process with a dual role in cancer progression. Here, we show that biglycan inhibits autophagy in two osteosarcoma cell lines (P ≤ 0.001), while rapamycin-induced autophagy decreases biglycan expression in MG63 osteosarcoma cells and abrogates the biglycan-induced cell growth increase (P ≤ 0.001). Rapamycin also inhibits ß-catenin translocation to the nucleus, inhibiting the Wnt pathway (P ≤ 0.001) and reducing biglycan's colocalization with the Wnt coreceptor LRP6 (P ≤ 0.05). Furthermore, biglycan exhibits protective effects against the chemotherapeutic drug doxorubicin in MG63 OS cells through an autophagy-dependent manner (P ≤ 0.05). Cotreatment of these cells with rapamycin and doxorubicin enhances cells response to doxorubicin by decreasing biglycan (P ≤ 0.001) and ß-catenin (P ≤ 0.05) expression. Biglycan deficiency leads to increased caspase-3 activation (P ≤ 0.05), suggesting increased apoptosis of biglycan-deficient cells treated with doxorubicin. Computational models of LRP6 and biglycan complexes suggest that biglycan changes the receptor's ability to interact with other signaling molecules by affecting the interdomain bending angles in the receptor structure. Biglycan binding to LRP6 activates the Wnt pathway and ß-catenin nuclear translocation by disrupting ß-catenin degradation complex (P ≤ 0.01 and P ≤ 0.05). Interestingly, this mechanism is not followed in moderately differentiated, biglycan-nonexpressing U-2OS OS cells. To sum up, biglycan exhibits protective effects against the doxorubicin in MG63 OS cells by activating the Wnt signaling pathway and inhibiting autophagy.

Explicit solvent repulsive scaling replica exchange molecular dynamics (RS-REMD) in molecular modeling of protein-glycosaminoglycan complexes.

Glycosaminoglcyans (GAGs), linear anionic periodic polysaccharides, are crucial for many biologically relevant functions in the extracellular matrix. By interacting with proteins GAGs mediate processes such as cancer development, cell proliferation and the onset of neurodegenerative diseases. Despite this eminent importance of GAGs, they still represent a limited focus for the computational community in comparison to other classes of biomolecules. Therefore, there is a lack of modeling tools designed specifically for docking GAGs. One has to rely on existing docking software developed mostly for small drug molecules substantially differing from GAGs in their basic physico-chemical properties. In this study, we present an updated protocol for docking GAGs based on the Repulsive Scaling Replica Exchange Molecular Dynamics (RS-REMD) that includes explicit solvent description. The use of this water model improved docking performance both in terms of its accuracy and speed. This method represents a significant computational progress in GAG-related research.

Molecular dynamics-based descriptors of 3-O-Sulfated Heparan sulfate as contributors of protein binding specificity.

Glycosaminoglycans are linear periodic and anionic polysaccharides found in the extracellular matrix, involved in a range of key biochemical processes as a result of their interactions with a variety of protein partners. Due to the template-less synthesis, high flexibility and charge of GAGs, as well as the multipose binding of GAG ligands to receptors, the specificity of GAG-protein interactions can be difficult to elucidate. In this study we propose a set of MD-based descriptors of unbound Heparan Sulfate hexasaccharides that can be used to characterize GAGs and explain their binding affinity to a set of protein receptors. With the help of experimental data on GAG-protein binding affinity, we were able to further characterize the nature of this interaction in addition to providing a basis for predictor functions of GAG-protein binding specificity.

Binding of heparan sulfate to human cystatin C modulates inhibition of cathepsin L: Putative consequences in mucopolysaccharidosis.

Mucopolysaccharidoses (MPS) are a group of rare lysosomal storage diseases characterized by glycosaminoglycan (GAG) accumulation causing progressive multi-organs dysfunction and ultimately severe cardio-respiratory damages. Human cystatin C (hCC), a potent inhibitor of cysteine cathepsins, plays an important role in respiratory diseases. However, its regulation remained unknown in MPS. Herein, elevated hCC levels were measured in respiratory specimens from MPS-I, -II, and -III patients and were significantly correlated with severe respiratory symptoms (rs = 0.7173). Heparan sulfate (HS), a prominent GAG, dampened its inhibitory activity toward cathepsin L in a dose-dependent manner. HS and HS-oligosaccharides bound tightly hCC, in combination with a secondary structure rearrangement. Molecular modeling studies identified three HS binding regions in hCC, including the N-terminus, which is crucial in the inhibition of cathepsins. Impairment of inhibitory potential of hCC may reflect abnormal regulation of proteolytic activity of cathepsin L in lung, ultimately contributing to the severity of MPS.

Impact of calcium ions on the structural and dynamic properties of heparin oligosaccharides by computational analysis.

Heparin (HP) belongs to glycosaminoglycans (GAGs), anionic linear polysaccharides composed of repetitive disaccharide units. They are key players in many biological processes occurring in the extracellular matrix and at the cell surface. GAGs are challenging molecules for computational research due to their high chemical heterogeneity, flexibility, periodicity, pseudosymmetry, predominantly electrostatics-driven nature of interactions with their protein partners and potential multipose binding. The molecular mechanisms underlying GAG interactions mediated by divalent ions, which are important for GAG binding to several proteins, are not well understood. The goal of this study was to characterize the binding of Ca2+ to two HP oligosaccharides of different lengths (dp10 and dp18, dp: degree of polymerization) and their impact on HP conformational space and their dynamic behavior with the use of molecular dynamics (MD)-based approaches with two Ca2+ parameter sets. MD data suggested that the flexibility of the monosaccharides, the glycosidic linkages and ring puckering were not affected by the presence of Ca2+, in contrast to H-bond propensities and the calculated Rg for a fraction of the oligosaccharide populations in both dp10 and dp18. Moreover, the essential differences in the data obtained by using two Ca2+ parameter sets were reported.